1. Field of the Invention
The present invention relates to a screening method for a drug target gene using chemical-genetic profile compendium of the heterozygous deletion fission yeast strain and the comparative genetic analysis using the same.
2. Description of the Related Art
According to the increasing interest in the quality of life due to the world-wide aging and the welfare society possibly made by the increase of general income of the people, the demand for medicine has been continually increased domestically and internationally likewise. Chronic diseases and new diseases are threatening human health these days. But, as basic science technology including bio-engineering techniques advances, studies on the diseases progress greatly, based on which the development of new medicine has also been advanced rapidly.
To develop a new drug, techniques of combinatorial chemistry and functional genomics have been used. Combinatorial chemistry technique is the method for screening an already known drug target through a library of organic-chemically synthesized compounds. In the method of functional genomics, animal cells or target cells are treated with a specific drug, which are then compared with the non-treated group by using DNA chip to identify a gene whose expression has been changed by that, in order to search a gene group related to disease or displaying a pharmaceutical effect. By taking advantage of the functional genomics technique, a mechanism responding to a drug and a target gene can be predicted, so that the acting point of the drug can be easily identified. The time and costs for the development of a new drug can also be saved and a cause of side effect can also be notified, enabling the prevention of it. However, this method has a disadvantage as well, which is it costs high expenses and requires more study to search a target gene.
In the meantime, the method of “chemical-genetic profiles” indicates the in vivo screening method to search a gene involved in the activation of a drug in cells by observing the changes of phenotype caused by genetic deletion based on the functional genomics after the cells have been treated with a specific drug. More precisely, changes of the sensitivity of heterozygous mutant to the specific drug are measured herein, by which almost every target gene can be easily identified. So, this method is useful for the screening of a target gene particularly in relation to a specific drug, a toxin, and a natural mixture.
In the method of chemical-genetic profiles, the genome-wide chemical-genetic profiles obtained from the deletion library of budding yeast like Saccharomyces cerevisiae (S. cerevisiae) are very useful for the identification of the unknown mode-of-action of a drug. In general, genome-deletion strain profiles facilitate the screening of a drug target gene or protein by screening a gene displaying a change in expression pattern by high-throughput spot assay (A. M. Smith et al., Pharmacol. Ther. 127 (2010) 156-164, A. B. Parsons et al., Nat. Biotechnol. 22 (2004) 62-69) and DNA-chip after treating target cells with a specific drug.
It has been reported that the success rate of target gene screening was increased to 70% from the previous rate of 25% when the screening of a target gene was performed with 80 conventional drugs by using the constructed budding yeast library (Lum P Y et. al., Cell. 2004; 116(1):121-137). According to the previous report, the principal of gene screening was based on drug-induced haploinsufficiency (DI-HI), that is the screening was achieved by measuring the reduction of such a gene that induced growth defect in the presence of the drug. According to the previous report, approximately 3% (180 genes) of the total 6,000 genes of a budding yeast displayed drug-induced haploinsufficiency even under normal culture condition (Deutschbauer et al., Genetics, 169 (2005), 1915-1925).
Schizosaccharomyces pombe (S. pombe) is a fission yeast, which belongs to ascomycetes like the budding yeast S. cerevisiae, but is not so closely related to S. cerevisiae in the evolutionary aspect. S. pombe (Wood V. et al., Nature. 45:871-880, 2002) is the 6th microorganism among the eukaryotes whose nucleotide sequence has been completely sequenced after S. cerevisiae (Goffeau A. et al., Science, 274:546-567, 1996). According to the sequencing result, S. pombe has the most efficient chromosome structure with less functional repeats, among the eukaryotes whose nucleotide sequence has been identified, and has the least protein determining genes, 4,824. However, 43% of the genes of S. pombe are known to contain intron. In addition, in S. pombe, such genes that are important in relation to cell cycle regulation, proteolysis, protein phosphorylation, and RNA splicing are well preserved. 31% of the genes of S. pombe are different from the genes of S. cerevisiae, and they are rather closer to those of human. Therefore, it is considered to be an efficient method to study the functions of S. pombe genes with comparing the genes of S. cerevisiae in order to understand the functions of human genes better.
Regarding the heterozygous deletion strain and the library thereof used for the chemical-genetic profiles, Korean Patent No. 10-0475645 describes method for screening of drug using systematic deletion mutant of fission yeast, and Korean Patent No 10-1098032 describes genome-wide construction of Schizosaccharomyces pombe heterozygous deletion mutants containing gene-specific barcodes by the methods of 4-round serial or block PCR, or total gene synthesis thereof.
Therefore, the present inventors tried to develop an efficient method for screening a drug target gene useful for the development of a new drug. As a result, the present inventors established the chemical-genetic profile compendiums of candidate drugs from the library of S. pombe heterozygous deletion fission yeast strain, and compared the profile compendiums with those of S. cerevisiae, the budding yeast, to select a drug target gene displaying sensitivity to the drug. At last, the present inventors completed this invention by confirming that the method for screening the gene targeting drug using the heterozygous deletion fission yeast strain of the invention could be efficiently used for the identification of a drug target gene in various eukaryotes.